Botrytis bunch rot (BBR) of wine grapes (Vitis vinifera), caused by Botrytis cinerea Pers., can result in significant financial losses for wine businesses through reduced fruit yield and inferior fruit composition for winemaking. Although infection of wine grapes may occur at any stage of fruit development, grape flowers are particularly susceptible to the establishment of latent infections in fruit. These infections arise when germ tubes produced from conidia of B. cinerea penetrate necrotic ‘scar’ tissue exposed when the calyptras or ‘caps’ fall from grape flowers. After penetration, the fungus remains in a quiescent phase inside the developing grape berry until BBR symptoms develop during fruit ripening. To prevent these infections in vineyards, protective fungicides are typically applied at the late-flowering stage when at least 50% of grape inflorescences have at least 80% of flowers in which the caps have fallen. To better understand the latent infection disease pathway and evaluate new crop protection solutions for managing BBR in grapevines, we have developed a reproducible system for establishing latent infections in planta. This system utilises propagated grapevines, producing inflorescences which can be inoculated with B. cinerea, and assessed for the incidence of latent infections under semi-controlled environmental conditions. We assessed the incidence of latent infections when grapevine inflorescences were inoculated with dry B. cinerea spores at early (less than 30% cap-fall) or late flowering (greater than 80% cap-fall). Inoculation during the late-flowering period resulted in twice the incidence of latent infections (45%) when compared with inoculation during early flowering (19%). Non-inoculated inflorescences resulted in an incidence of latent infection of 7%, likely due to background environmental contamination. Using this system, we present data on Switch® fungicide efficacy in relation to the timing of inoculation and fungicide application. This system will also provide a valuable tool for evaluating new crop protection solutions for BBR, bridging the gap between in vivo fungicide studies and field trials. Our work forms part of a broader platform to evaluate the efficacy of new RNA-interference based crop protection solutions, including BioClay™, for control of botrytis diseases in wine grapes and other crops.